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Leghemoglobin

Introduction

Leghemoglobin is a protein that is required by Legume plants in order to fix nitrogen by participating in the Nitrogen Fixation pathway. It is found in legumes such as cowpea, soybeans, alfalfa, and other different types of beans. Leghemoglobin functions by keeping the oxygen at a specific low concentration to the bacteroids to allow for respiration, but at the same time, this low concentration keeps the oxygen out of the nitrogen cycle so it doesn’t inhibit the nitrogenase activity (Ref 1).

Leghemoglobin is a 16 kD globin protein containing eight secondary alpha helices, constituted by a heme group(protoporphyrin XI) and a single polypeptide. In its ferrous (reduced state), Leghemoglobin binds oxygen for transportation. (Ref 5). It is hypothesized that Leghemoglobin can be formed by one of the two ways:

1. The Rhizobium bacteroid (Ref 2) is thought to synthesize the heme group in the root nodules of the legume, where it then gives the plant the heme to complete the synthesizing of the Lb (Ref 1).
2. The plant itself has also been thought to possibly produce the heme itself, in the mytochondria of the plant cells. It then combines with the peptide sequence to complete the whole protein (Ref 1).

Function

In order to fix nitrogen, legume nodules require Leghemoglobin. In 1958, Bergersen and Appleby showed that only the ferrous form of Leghemoglobin binds to oxygen in the cytoplasm of the infected plant cells. (Ref 1). Therefore, the function of Leghemoglobin is to carry the oxygen for the bacterial respiration, as well as for nitrogen fixation.

The enzyme needed for nitrogen fixation is nitrogenase. However, this enzyme is inactivated by oxygen, but at the same time, oxygen is needed for the bacteria to reduce nitrogen to ammonia. This ambiguity is explained by the function of Leghemoglobin, which transports oxygen at a low but stable concentration allowing for the symbiotic operation of nitrogenase activity and bacterial respiration.

Structure

The 16 kDa polypeptide leghemoglobin consists of two main subunits, the main globin structure and the iron-encompassing heme (protoporphyrin XI) group. These two subunits form a molecule structurally similar to Myoglobin (Ref 1). The globin fold portion of the molecule is the standard globin secondary structure, a series of 8 alpha helices (Ref 5). Depending on the species and specific type of Leghemoglobin, the primary amino acid sequence can differ some, but overall it is well conserved.

The heme prosthetic group consists of four 5-membered pyrrole rings, forming a cyclic ring around a central iron (Fe) atom. This atom is contained within four equatorial nitrogens, in addition to another nitrogen from a close histidine residue and an opposite dioxygen (Ref 9). The ligand contact residues along the heme group are Phe30, His63, His97, and Val67. Depending on the specific type of Leghemoglobin, some of these specific residues are disordered and others interconvert between two conformations (Ref 7).

In opposition to the differences in polypeptides among globin proteins (Hemoglobin and Myoglobin), the heme group has been found to remain largely the same (Ref 1). The main difference is a considerably larger heme group in Leghemoglobin than its other oxygen-transferring counterparts. The way the heme group attaches to the polypeptide is also different. The steric crowding around the ligand binding site (beside the heme) is reduced, plus there is an altered packing at the proximal side of the heme and conformational differences along the distal side. As a result, the oxygen affinity is larger than that of Myoglobin and Hemoglobin (Ref 8).

The methods by which various Leghemoglobins were purified, and then analyzed, are as follows:

• Ammonium Sulfate Precipitation
• Concentration by Ultrafiltration
• Electrophoresis
• Anion-exchange Column Chromatography
• Isoelectrofocusing
• Nuclear Magnetic Radiance (NMR)
• X-ray crystallography (Ref 1 and 6)